The present invention relates to a novel S6 kinase (p70xcex2Sk6), mutant variants thereof methods of making and using this S6 kinase, and related nucleic acids and Ski antibodies. The invention also relates to binding partners of the S6 kinase, methods of identifying the binding partners and antibodies thereto.
The 40S ribosomal protein S6 is a component of the 40S subunit of eukaryotic ribosomes. The ribosomes are part of the cellular machinery responsible for translation of mRNA and protein synthesis. The S6 protein is phosphorylated in response to certain cellular signaling events such as hormone or growth factor induced cellular proliferation. p70 S6 kinase (p70S6k) is responsible for S6 phosphorylation and is believed to be the major physiological S6 kinase in mammalian cells (Proud, 1996 Trends Biochem. Sci. 21:181-185).
1. p70xcex1 S6 Kinase
A. Structure and Function
The first p70 S6 kinase identified was the alpha (xcex1) form. The gene encoding the human p70xcex1 S6 kinase (70S6k)was isolated in 1991 (Grove et al., 1991 Mol. Cell. Biol. 11:5541-5550). Other p70xcex1 S6 kinase sequences have been described in Mus musculus (GenBank Accession No. SEG_AB015196S, AB015197, and AB015196), Xenopus laevis (GenBank Accession No. X66179), and rat (GenBank Accession No. M57428).
Two p70xcex1 S6 kinase isoforms were identified: p70xcex1-I GenBank Accession No. M60724) and p70xcex1-II (GenBank Accession No. M60725). The two p70xcex1 S6 kinase isoforms differ only in their amino tenmini by 23 amino acid residues resulting in a 70 kD protein and a 85 kD protein. The isoforms are referred to in the literature as p70S6k/p85S6k or p70xcex1 S6 kinase. Both isoforms share similar activity towards ribosomal protein S6 in vitro but are expressed in different cells and tissues. The two isoforms are produced by two mRNA products and are not a result of post-translational modifications. They are serine/threonine kinases and are known to act on the substrate KKRNRTLSVA (SEQ ID No. 7) (Pai et al., 1994 Eur. J. Immunol. 24:2364-8; and Leighton et al., 1995 FEBS Letters 375:289-93).
The p70xcex1 S6 kinase plays an important role in the progression of cells from G1 to S phase of the cell cycle and in the initiation of protein synthesis. Recently, p70xcex1 S6 kinase has been demonstrated to regulate the translation of a class of mRNAs containing an oligopyrimidine tract in their 5xe2x80x2 untranslated region. This class of mRNAs, termed 5xe2x80x2TOP mRNAs, represent up to 20% of the a cell""s total mRNA. Many of the proteins encoded by 5xe2x80x2TOP mRNAs are translational apparatus proteins and cell-cycle progression proteins.
The p70xcex1 S6 kinase has four identified interdependent domains: (1) a catalytic domain, (2) a kinase extension domain, (3) a pseudosubstrate autoinhibitory domain, and (4) the N-terminal domain. The catalytic domain is located in the middle of the protein and is followed by the kinase extension domain, which is a unique feature for the PKA family. The pseudosubstrate autoinhibitory domain is also unique for the p70xcex1 S6 kinase, not having been observed in any other known kinases. It possesses 5 phosphorylation sites which are responsible for the p70xcex1 S6 kinase regulation. The N-terminal domain mediates the sensitivity for rapamycin, which strongly inhibits serum-induced phosphorylation and activation of the p70xcex1 S6 kinase. This domain may also mediate the interaction with a yet unknown phosphatase.
B. Regulators and Cascades
Growth factors, such as insulin, and mitogens are known to activate in vivo p70xcex1 S6 kinase (Alessi et al., 1998 Curr. Biol. 8:69-81). Heat shock also activates p70xcex1 S6 kinase (Lin et al., 1997 J. Biol. Chem. 272:31196-31202). Certain drugs have been identified that regulate p70xcex1 S6 kinase activity including: rapamycin, wortmannin, Ro31-8220, GF109203X, LY294002, phenylephrine (PE), PD098059, SQ20006, polymerized collagen, forskolin, interleukin-10 (IL-10), demethoxyviridin, phorbol 12-myristate 13-acetate (PMA), A23187, bombesin and antibodies which recognize the p70xcex1 S6 kinase (Proud, 1996; Morreale et al., 1997 FEBS Letters 417:38-42; Kanda et al., 1997 J. Biol. Chem, 272:23347-23353; Boluyt et al., 1997 Circ Res. 81:176-186; Coolican et al, 1997 J. Biol. Chem. 272:6653-6662; Koyama et al., 1996 Cell 87: 1069-1078; Busca et al., 1996 J. Biol. Chem. 271:31824-31830; Crawley et al., 1996 J. Biol. Chem. 271:16357-16362; and Petritsch et al., 1995 Eur, J. Biochem. 230:431-8). The immunosuppressant rapamycin (Rap) is the most potent inhibitor of p70xcex1 S6 kinase described (Pullen et al., 1997 FEBS Letters 410:78-82).
p70xcex1 S6 kinase is an enzyme which lies downstream of phosphoinositide 3-kinases (P13-kinase). The mechanisms regulating the p70xcex1 S6 kinase have not been fully elucidated. P13-kinase has recently been shown to activate another phosphoinositide-dependent protein kinase, termed PDK-1. So far, only PDK-1 has been shown to phosphorylate p70xcex1 S6 kinase in vivo, and this phosphorylation is essential for p70xcex1S6k activity towards ribosomal S6 protein. Wortmannin, a fungal inhibitor which down-regulates the p70xcex1 S6 kinase, is believed to act by inhibiting PI-3 kinase. In contrast, another fungal inhibitor, rapamycin, inhibits the p70xcex1 S6 kinase by another cascade pathway involving the mammalian target of rapamycin (mTOR; also known as RAFT or FRAP) (Proud, 1996; Stewart et al., 1994 BioEssays 16:809-815). mTOR is a member of the PIK-related family of protein kinases (Pullen et al., 1997). Additional regulators of the p70xcex1 S6 kinase include, but are not limited to protein kinase B (PKB), Cdc42, and Rac. The role of most of these proteins as p70xcex1 S6 kinase regulators has yet to be fully elucidated.
The present invention is based on our discovery of a new gene which encodes a novel S6 kinase (p70xcex2S6k). The invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, (e.g., SEQ ID No.1) an isolated nucleic acid molecule that encodes a fragment of SEQ ID No.2, an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID No.1 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2 under conditions of sufficient stringency to produce a clear signal.
The present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with the nucleic acid molecule of the invention under conditions in which the protein is expressed.
The invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID No.2, an isolated polypeptide comprising a fragment of SEQ ID No.2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID No.2 and naturally occurring amino acid sequence variants of SEQ ID No.2.
The invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies and fragments thereof.
The invention further provides methods of identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2.
The invention further provides methods of identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the protein to the agent; and determining whether the agent modulates at least on activity of said protein, thereby identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2.
The invention further provides methods of identifying binding partners for a protein comprising the sequence of SEQ ID No.2 or activated variants thereof, comprising for example, the steps of: exposing said protein to a potential binding partner, and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a protein comprising the sequence of SEQ ID No.2. Exposing may be accomplished by expressing the protein in a cell.
The present invention further provides methods of modulating the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2.
The invention also provides methods of modulating at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2.